B cells provide us with antibodies against a multitude of infectious microorganisms. In humans, B cells are produced in the bone marrow throughout life in a process called B lymphopoiesis. Even so, in human, B cell immunity declines with age leaving the elderly more susceptible to infections and autoimmune diseases, and less responsive to vaccinations. In rabbits, B lymphopoiesis occurs for only a few weeks after birth, and yet, they maintain robust B cell immunity throughout life. In this grant, we will study the mechanism by which B lymphopoiesis is developmentally-terminated in rabbits;whether gut-associated lymphoid tissues (GALT) contribute to maintenance of B cell homeostasis throughout the rabbit's life in the absence of ongoing B lymphopoiesis;and how B lymphopoiesis can be reinitiated. In Aim 1, we will identify and characterize the stage of differentiation at which B lymphopoiesis is arrested in bone marrow by using in vitro cultures and adoptive transfer of putative lymphocyte progenitors. In Aim 2, we will determine the turnover rate of B cells in GALT by incorporation of BrdU, and determine the rate at which GALT B cells seed other peripheral lymphoid tissues. Further, we will assess the contribution of GALT to maintenance of B cell immunity by surgically removing all organized GALT from adult rabbits and testing for the first time in any species, whether GALT serves as a reservoir of B cells for other lymphoid tissues throughout life. In Aim 3, we will use antibodies and in vivo expression of decoy soluble receptors or extracellular matrix molecules to test if one or more of three candidate molecules, activated Notch-1, a splice variant of IL-7, or an extracellular matrix protein, has the capacity to reinitiate B lymphopoiesis. Data from these experiments will provide an understanding of the mechanism by which B lymphopoiesis declines, and how B cell immunity can be maintained in the absence of de novo development of B cells. These results should provide potential solutions for maintaining robust immune responses in the elderly as B cell immunity declines. The research proposed in this grant is significant because by the year 2030, 20% of the US population is expected to be age 65 or older and have diminished B cell immune systems. This work has the potential to identify targets for therapeutic interventions that will allow the elderly to maintain a healthy immune system.